Methods for producing streptomycin and vitamin b12



Patented Oct. 20, 1953 METHODS FOR raonuome STREPTO- .MYCINAND VITAMIN B12 I R.obert- B."lMciilormaek and- Asger F. 'Langlykke, HighlantL'Parkand David Perlman," Princeton, Q-N, .L, assignors, :by mesne :as'si-gnments, to iMathiesom-fihemical Corporation, Baltimore, -Md.,za corporation-of Virginia No- Drawing. fApplicatiomFebruary 3, 1949,

P Serial- NoJ'M-ASZ (CL: 195-v-80') Thisinvention relates to methodsoiiproducing medicinally-usefu-l substances (inter -aalia,--antibioticsand; vitamins) by culturing or anisms.

: Prior to. this inventiongit :had been shown that a potent antibiotic, called streptomycin ispresent in the culture liquid obtained-when asuitable stra'in of the organism fitreptomyces -gri -seus is grown in i or on certain liquid: nutrient media (Schatz', 3ugie, and Waksman; -Proc. Soc. Exp. Biol; and Med; 1944,55, 6.6) ;-t and that the solids (the mycelia, spores, -:and other vsedimentable solids) of. 1 a culture of Streptomyces A griseus that has produced streptomycin contain additional streptomycin, which may i be obtainedtby. subjecting these solids to extraction-wither: aqueous .acid, andrecovering the streptomycin inwthe-ex- "tract (application SeriakNoofiSfifiZ l; filed-.iApril '5 1-945,' now 'Patent' N o. 2,461,922, dated=- February 15, 1949.). .7 [The term extractiontiszusedherein its. broad sense, and: hence comprehends elution; is: e.,\ the separation =(e.; g of adsorbedmaterial) by washing] I Although such acid-extraction bf the culture sriliiis is highly advantageous in that tit; provides van additional yield of streptomycin (Tori enhances the -activity of the streptomycin-containing cultureliquid), the reactivity with the 'extraction equipment thereby introduced necessitates the .use of acid-proof equipment. 1 I

- It is the object of. thisf'invention-to provideimproved methods of recovering the streptomycin inthe solids of a culture of Streptomyces: g'riseus that has produced streptomycin.

The methods of thisinvention-essentially comprise-subjecting'the solids of a culture of- Streptomy'ces griseus that has produced streptomycin to "extraction with an aqueous medium whosecon- :centration of. highly' dissociatable'esaltssissubstantia'lly greater than ithat of the culture liquid,

- and recovering the streptomycin in :the extract. [By '-c"ulture of Streptomyc'es griseusthat has produced streptomycin is meant; of course; the culture obtained by growing astreptomycin-producingstrain of Streptomyces griseus under 'conditions and in a medium suitable'iior'the production of streptomycin] The extraction for, the s'olids. may. be efie'cted eitherwhile they are still inht-he culture on afteretheir separation .iromfthe culture liquid, or at both stages.

a Thus, the extraction may be reflected Wedding 1 a: highlyedissociatable usaltw to the :(w-holebculture fiafterithe fennentationt-has pro'ceededmo the. de-

-'sired'- 'extent; genenally:v untilnsubstantially+maxi- :mum istreptomycin :productivity has been :reached] ;:and intimately contacting ihelsolid and sliquid: components :of: the culture. in this case, the streptomycinin, the: solidsqis extractedt into the streptomycin-containing liquid; and i on separation-of the solids by centrifugation and/or filtration. 'ca strentomycin-e-containing :culture liquid (-A):zof=enhancedt activityi'is obtained. =-A1- ternatively; the-solids: are first: separated from the culture liquid; in which case the filtrate (13): corresporids to the streptomycin-containing culture liquid obtained prior to use oi' the' acid-extraction invention the separated solids (which may 'first'be washed with water, the-washings being with an \aqu'e011SLmediumcontaining a. highly- :dissociatablexsalt: to --ob.tain--.a,nextract: .(A1 )2 l mtaining additional-e'streptcmycin. vThe pneDhrations A and-3B1,-as well'as'preparationzsBc (separately, or combined with"B1) and Aiiseparately, or- 'combine rwith-A) may be used -or further treated as 'primary -streptomycin-containing -liquids (e g.,*treated-to' purify andrecover'the streptomycin and/or a'derivativethereof;epgq

'dihydrostreptomycin) and/orto recoverthe vitamin B12 content. thereof.

:The; natureof the .highly-dissociatablensalt employed does-not have a qualitative Jefiect on the extraction, provided, of course, that thesaltdoes not; precipitate or inactivate the streptomycin; preferablypthesalt-:is at water-soluble, alkali- -metal (including-'ammonium) or, alkaline-earth 1 metalx salt or a: lovwmolecular-weight acid,- such sodium "citrate; "sodium nitrate; sodium sulfate, ammonium chloride, ammonium -tartra-te,:=. am-

' monium sulfate, potassium chloride, calcium "chloride, 'calciumsulfate, barium chloridepmagnesium sulfate, ammonium acetate; and-aluminum potassium su f e The concentration oflthesalt required to effect .maXi-mum extraction of strep mycin fro "th culture :1 sq idskis gene ally less' h n. about: 1.0 molar. Thus, the salts listed1 hereinafter areeffective in obtaining maximum recovery in the molar concentrations indicated:

NaCl v1.0

NI-I4C1 0.1 Ammonium tartrate 0.1

Na2SO4 0.05

KCl 0.8

MgSOMHzO 0.025

BaClz -1 0.1

CaClZ 0.025

The salt-extraction of this invention (like acid-extraction) facilitates separation of the culture solids by centrifugation, because it promotes coagulation of the solids. The salt-extraction may, of course, be combined with the acid-extraction priorly used, the presence of acid facilitating filtration of the extract as well as lowering the concentration of the salt required for maximum extraction. [Filtration of the extract is facilitated also by use of a filter aid (e. g., a diatomaceous-earth filter-aid such as Celite), both with salt and salt-plus-acid extraction] Actually, relatively slight pH adjustment in either direction (from the normal pH of the culture liquid at the end of the fermentation), e. g., by addition of sulfuric acid or sodium hydroxide, enhances the effectiveness of a low concentration of the salt in extracting the anti biotic. Furthermore, mixtures of salts may be used. The salt, or mixture of salts, or mixture of salt and acid may be added in undissolved or aqueous-solution 'form (preferably concentrated).

The primary streptomycin-containing liquids obtained in accordance with this invention may advantageously be purified and/or further treated as follows-preferably after partial purification by treating the primary streptomycin-containing liquid (desirably, after being substantially neutralized, if not already so) with an activated charcoal or a cation-exchange resin of the carboxylic-acid type (such as IRCOso) and eluting the streptomycin from the adsorbent with an aqueous, water-soluble mineral acid:

1. The impure streptomycin-containing liquid is intimately contacted with a surface-active agent of the organically-substituted polybasicinorganic-acid type and a substantially waterimmiscible organic solvent for soaps; the organic solvent phase is recovered; and the salt-type derivative of streptomycin therein is converted into a water-soluble salt of streptomycin. [This purification method is the subject of Lott, Bernstein and Heuser application Serial No. 767,851, filed August 9, 1947, now Patent No. 2,537,933, dated January 9, 1951.]

1alternative. The impure streptomycin-containing liquid is treated with a surface-active agent of the organically-substituted polybasicinorganic acid type; and the insoluble salt-type combination of streptomycin and the surfaceactive agent is recovered, and converted into a water-soluble salt of streptomycin. [This purification method is the subject of Lott, Bernstein and Heuser application Serial No. 767,852, filed August 9, 1947, now Patent No. 2,537,933, dated January 9, 1951.]

2. The impure streptomycin-containing.liquid, or an aqueous solution of purified streptomycin obtained as described in Section 1 or l-alternative hereinbefore, is treated with hydrogen in the presence of a hydrogenation catalyst to obtain dihydrostreptomycin.

It has been found that the salt extract B1, as well as the corresponding acid extract [i. e., the extracts obtained from solids separated from the culture without prior addition of a salt and/or acidification] may contain about half of the streptomycin in the culture, and that their watersoluble-impurity content is relatively low, as compared with filtrate B; also, that a higher recovery of streptomycin is obtained when the solids are extracted after separation from the culture liquid. Accordingly, it may be advantageous to filter, then extract, and treat filtrate B and extract B1 separately. By treating extract B1 separately (or the similar extract obtained by acid-extraction), the carbon-adsorption step may be eliminated, there being a high loss (generally about 40%) on purification by carbon adsorption and elution. Moreover, the relatively-low watersoluble-impurity content of extract B1, (or the similar extract obtained by acid-extraction) makes possible the direct conversion of the streptomycin therein to dihydrostreptomycin and purification of the dihydrostreptomycin formed (instead of first purifying the streptomycin and then converting it to dihydrostreptomycin).

Vitamin B12, which is formed along with the streptomycin (McCormack application Serial No. 53,761, filed October 9, 1948), also is extracted from the culture solids by the salt-extraction; and in this connection, salt-extraction is advantageous over acid-extraction, because part of the vitamin B12 formed is in acid-precipitable form (and hence is not recoverable from the acid extracts). Thus, liquids A, B, and B1 may be treated to recover the vitamin B12 as the primary product [for example, by extraction with 88% phenol or by treatment with an activated charcoal which is a good adsorbent for vitamin 1312]; or these liquids may be treated to recover the streptomycin as the primary product [for example, by treatment of the liquid with an activated charcoal or cation-exchange resin which is a good adsorbent for streptomycin, and elution of the streptomycin] and to recover the vitamin B12 as a by-product [in the charcoal treatment, by elution of the charcoal (after removal of streptomycin) with aqueous acetone or aqueous isopropyl alcohol, for example; and in the resin treatment, by treatment of the liquid (after removal of the resin and adsorbed streptomycin) with an activated charcoal, for example, which adsorbs vitamin B12, and elution therefrom].

Although the extraction of streptomycin from the culture solids, as measured by bioassay, rises to a peak and then falls as the concentration of the salt is increased (especially on extraction with salts of polyvalent ions, e. g., magnesium sulfate, ammonium sulfate, calcium chloride and sodium sulfate), chemical assay indicates the drop in streptomycin titer to be apparent rather than real, i. e., to be due to the effect of the salt on the bioassay. The optimum concentration of any particular salt is readily determinable, being the lowest concentration capable of effecting substantially complete extraction of the streptomycin in the solids of the culture.

Salts (especially sodium chloride) are often included in the medium in which the Streptomyces griseus is grown for the production of streptomycin, being added separately and/or contained in the soybean meal or other medium component; and a highly-dissociatable salt may therefore be present in the culture liquid at the end of the fermentation. However, such salt is not present in sufiicient concentration to effect subassume staiitia 'eittfistiom of the stfeptomychi iri the myceli um; ai'id th nitial inclusiori' iifsuflicient salt to efict such extraction 'woiild lnhibit the production Iof streptomycin as well as incre'aise the'corrosion of the fermentation equipment.

The oilowi'ngexampies are illustrative of the invention:

a. -1100=ga1of an'aqueous ('tapwater) -=medium containing 2.25 soybean meal and 1.62 .-hydrated dextrose (es g., Cerelose) is-placedim a 1300-ga1. carbon-steel fermentation tank equipped with a -stirrer and air sparger;- and' the tankis sterilized for'one" hour 'at l20 C. When the-temperature reaches 25 0.;50 gal. "ofa-strptomycinproducing strain or St-rem)omyees orz'seus inoculum is added and the 1 inoculated medium -is incubated at'-25 C; under:- a pressure of 3-6 lbs.

pensquare inch "(an average of l cu. it; air/gal.-

medium /hour being passed throughthe medium) while stirring (at al'rate of 120 :R. P. M.) During the incubation; 5.6 liters of lard oil is" added uno wise as an antifoam agent.

bi 0.12 gJ MgSO4-7H2O is 'added to ml. of a two-day old culture" prepared as'describedin a (the nitrate of 'which"has a potency "of 183 uf1-its/ml.-,=and a pH of 7.3) i=-andthe-mixture is well shaken for lid-minutes;-centrifuged,+and the supernates'eparated. The supernate; which has a potency of about 275 units/ml, is-used or further treated as a streptomycin coritaining culture filtrate, e;' g., treated to purify and 'recover the streptomycin and/or convertthe strleptomycin to dihydrostreptomycin, -ahd'(if-=d'esired) to recover vitamin B12 as a "by-product.

Example .2

9.5- g5 sodium sulfate is 'addedto'*250'-m1:*of a 41-hour "old streptomycin-containing culture 'lp' repar'ed, for example," as" -des'cribedin I a "of Example i) the" filtrate-of which has a potency of 15711111133/1111'. and a of 6.35; ahdfthhiixture' is agitated thoroughly for-30 minutes; and filtered. The filtrate has a-streptor'nycimpotency ofabout 325- units/ml.

Example 3 To a streptomycin-containing culture having a potency of "138 units/ml.--and-a 'of -5.95"is added sufficient sodium chloride and sodium hydroxide to-make the cultureliquid at 0.1;mol'ar and" 0.001 'molar' solution thereof; respectively, the.. being about 6.7; an the mixture is agitated thoroughly ahdfilt'e'r'ed. "The filtrate has a streptomycin =-potency of about 192 units/ml.-, as-contrastedwith-practically no enhancement of potencyon-adding sodium hydroxide to molar concentrations as high as 0.012 without the sodium chloride.

: Example 4 To a ""Streptomycin-contiihihg culture' h'ai iin'g a potencyof 1'3s"units7m1; arid a" pH 'of 5'l95is added sufliicient sodium chloride A and V sulfuric acid to makefthe culture liquid a 0.1 molarand 0.()35"1i-1olar solution thereof, re'sp'ectivelyfthe 'pI-I dieing about 2.05 and thehriiithreisf eigi tated thoroughly and filtered. The filtratahas a streptomycin potency of about 218 units/ml, as contrasted with an enhancement of potency to only about 184 units/ml. on adding sulfuric acid to a molar concentration of 0.031 without the sodium chloride.

6" V .mtample: 5

nrolar sodium chloride solution].

Example 6 two-day old streptomycin-containing culture (prepared for example, as describedinsection a of Example-l) isfiltered. The filtrate, having a potency of about 180 units/ml., is further treated in the conventional manner to purifyand'recovefed thef streptomycin'therein and/or tdcohvert itto dihydrostreptomycin.

The solids filtered oil are extracted with a volume of 0.2 molar sodium chloride solution equal to the'volume' of the culture, and filteied. [This filtrate, having a 'p0ten'cy "of {"about "140 units/ml, is treated either separately or together with the 'original' 'c'ulture filtrate to purify and recover the streptomycin and/or convertthe streptomycin -todihydrostreptomycin. Thetotal streptomycinrecovery thus obtainable exceeds that obtainable by ""acid-e'iitraction of the culture.

Essentially the same recovery of" streptomycin from 'the"separated culture solids is obtained 'by using a 0.1"nic'il2'u solution "of Sodium sulfate or a 0.05 molar solutionhff'fr'riagriesium sulfateiri place of the 0.2"mo1ar solution of sodi'um' 'chl'or'idef u'1o1 "p'I-I adjustment is substantially tvithout 'efict' in extracting the separated culture'solids with the salt solutions.

"Extraction "of theseparfated culture solids with af'voliimeofthe salt solution (or acid solution) substantially less'thari thevolume of th'cul'ture liquidserves as a meansof obtaining a'relatively concentratedsoiution of streptomycin.

Example 7 'lgo astreptomycincontaining culture having apctericy "of units/intend apH of 7.3"Was added sufficient sodium sulfateto'make'the'culture liquid a 0,1 momrsqmticntherecr; and the enhancement "of" potency "determined at various pH levels; the results were as follows '[the "potericy ofthe' culture at the same pl-ideve'lswithoiit the*addition'ofsodiumsulfate being given for comparison] As indicated by these results, extraction by a 0.1 molar sodium sulfate solution is virtually unaffected by pH; and conversely, pH adjustment may be made for such purposes as ease of filtration without materially affecting the salt extraction.

Example 8 To a three-day old streptomycin-containing culture prepared as described in a of Example 1 except for the inclusion of 1 mg./liter of CC12'6H2O in the medium to enhance vitamin B12 production is added sufiicient MgSO4'7I-I2O to make the culture liquid a 0.05 molar solution thereof; and the mixture is agitated thoroughly and filtered. The filtrate, having a vitamin B12 content of 0.18 gamma/ml, may be treated as described in the aforementioned McCormack application to recover the vitamin B12 as the primary product or as a by-product of streptomycin.

Similar salt-extraction with the following salts in the concentration indicated yields filtrates of the indicated vitamin B12 content:

Salt-extraction with the same salts and in the same molar concentrations may be applied to the solids which have been separated from the culture liquid without treatment, yielding filtrates of high vitamin B12 (as well as streptomycin) content.

The salt-extraction of this invention is of general utility, being applicable to other species of Streptomyces which produce antibiotics. Thus, the solids of a culture of Streptomyces venezuelae that has produced chloramphenicol [Science 106:417 (October 31, 1947)] contain additional chloramphenicol, which may be obtained by subjecting these solids to salt-extraction, and recovering the chloramphenicol in the extract; the procedure being fully analogous to that described hereinbefore for extraction of streptomycin from the solids of a culture of Streptomyces grz'seus that has produced streptomycin.

The invention may be variously otherwise embodied within the scope of the appended claims.

We claim:

1. The method of producing streptomycin, which comprises culturing Streptomyces grz'seus in contact with a suitable liquid nutrient medium to produce streptomycin, adding a highlydissociatable salt to the culture, intimately contacting the solid and liquid components of the culture, and separating the liquid component from the solid component.

2. The .method of producing streptomycin, which comprises culturing Streptomyces griseus in contact with a suitable liquid nutrient medium to produce streptomycin, separating the solids from the culture, subjecting the solids to extraction with an aqueous medium whose concentration of highly-dissociatable salts is substantially greater than that of the culture liquid, and separating the extract from the solids.

3. The method of producing streptomycin, which comprises culturing Streptomyces griseus in contact with a suitable liquid nutrient medium to produce streptomycin, subjecting the solids of the culture to extraction with an aqueous medium whose concentration of highly-dissociatable salts is substantially greater than that of the culture liquid and whose pH is either side of that of the culture liquid at the end of the culturing, and separating the extract from the solids.

4. The method of producing streptomycin, which comprises culturing Streptomyces griseus in contact with a suitable liquid nutrient medium to produce streptomycin, subjecting the solids of the culture to extraction with an aqueous medium whose concentration of highlydissociatable salts is substantially greater than that of the culture liquid and whose pH is lower than that of the culture liquid at the end of the culturing, and separating the extract from the solids.

5. The method of producing streptomycin, which comprises culturing Streptomyces grz'seus in contact with a suitable liquid nutrient medium to produce streptomycin, separating the solids from the culture, subjecting the solids to extraction with an aqueous medium whose concentration of highly-dissociatable salts is substantially greater than that or" the culture liquid and whose volume is substantially less than that of the culture liquid, and separating the extract from the solids.

6. The method of producing streptomycin, which comprises culturing a streptomycin-producing strain of Streptomyces grz'seus in submerged culture in a liquid nutrient medium essentially comprising soybean meal and dextrose, subjecting the solids of the culture to extraction with an aqueous medium whose concentration of highly-dissociatable salts is substantially greater than that of the culture liquid, and separating the extract from the solids.

7. In the method of producing a Streptomyces griseus metabolite of the group consisting of streptomycin and vitamin B12, the steps of culturing Streptomyces griseus in contact with a liquid nutrient medium suitable for the production of said metabolite, subjecting the solids of the culture to extraction with an aqueous medium whose concentration of highly-dissociatable salts is substantially greater than that of the culture liquid, and separating the extract from the solids.

ROBERT B. MCCORMACK. ASGER F. LANGLYKKE. DAVID PERLMAN.

References Cited in the file of this patent UNITED STATES PATENTS Name Date Rake Feb. 15, 1949 Number OTHER REFERENCES 

7. IN THE METHOD OF PRODUCING A STREPTOMYCES GRISEUS METABOLITE OF THE GROUP CONSISTING OF STREPTOMYCIN AND VITAMIN B12, THE STEPS OF CULTURNING STREPTOMYCES GRISEUS IN CONTACT WITH A LIQUID NUTRIENT MEDIUM SUITABLE FOR THE PRODUCTION OF SAID METABOLITE, SUBJETING THE SOLIDS OF THE CULTURE TO EXTRACTION WITH AN AQUEOUS MEDIUM WHOSE CONCENTRATION OF HIGHLY-DISSOCIATABLE SALTS IS SUBSTANTIALLY GREATER THAN THAT OF THE CULTURE LIQUID, AND SEPARATING THE EXTRACT FROM THE SOLIDS. 